PROJECT SUMMARY/ABSTRACT - Project 1 An important regulator of cell physiology in eukaryotes is the eight-protein exocyst complex, which traffics subsets of intracellular vesicles for exocytosis to particular sites on the plasma membrane. Recent studies of mammalian epithelial cells have shown the exocyst is critical for several aspects of differentiation and morphogenesis, such as epithelial barrier integrity, cyst and tubule lumen formation, and assembly and signaling of primary cilia. However, what remain poorly understood are the molecular mechanisms by which epithelial cells direct the exocyst to accomplish so many different functions. Identifying and characterizing these mechanisms will be central to better understanding the development and physiology of mammalian epithelial tissues. We have recently established novel model systems to investigate the exocyst's regulation of cellular trafficking and tissue development, including the first tissue-specific exocyst conditional knockout mouse model. From our studies of the exocyst during renal development, and in collaboration with an Institute of Biogenesis Research COBRE-1 Project Leader, we have identified interesting parallels and variances of exocyst regulation during renal cystogenesis and pre-implantation blastocyst cavitation. Based on our preliminary findings, our hypothesis is that epithelial cells, at each stage of differentiation, display an array of regulatory mechanisms to temporally and spatially control exocyst-mediated trafficking, which is vital to normal epithelial tissue morphogenesis. We will test this hypothesis through the following Specific Aims: (1) Identify epithelial-specific regulatory mechanisms that redirect exocyst-mediated trafficking during mesenchymal-to- epithelial transition (MET). Here we use an induced pluripotent (iPS) cell model of MET, and in vivo models of MET during blastocyst formation and kidney development, to evaluate modifications of the exocyst that may induce the specificity of epithelial cargo and trafficking. (2) Determine if the exocyst directs the polarized exocytosis of solute carriers and aquaporin channels necessary for unidirectional fluid transport into growing epithelial lumens. We will use our novel exocyst conditional knockout mice, and cell lines established from these mice, to test if the exocyst mediates translocation of key solute transporters and aquaporin channels during cystogenesis, comparing differences during blastocyst cavitation and renal vesicle formation. (3) Identify the biochemical interactions that control the exocyst's trafficking to primary cilia in polarized epithelial cells and their importance to blastocyst morphogenesis. We will investigate which of the eight exocyst subunits localize to the cilia, test which are necessary for ciliogenesis, and screen for exocyst-binding proteins specifically in the cilia. We will also investigate the exocyst's role in primary cilia trafficking in the blastocyst. The anticipated outcome of the project is characterization of new genetic and biochemical mechanisms that mammalian epithelial tissues use to control polarized exocytosis during their differentiation, growth, and morphogenesis.